U.S. patent application number 15/536798 was filed with the patent office on 2017-12-07 for method and measuring device for determination of the growth rate of biofilm.
This patent application is currently assigned to Helmholtz-Zentrum fuer Infektionsforschung GmbH. The applicant listed for this patent is Helmholtz-Zentrum fuer Infektionsforschung GmbH. Invention is credited to Mark BROENSTRUP, Blanka KARGE, Jost VAN DUUREN, Christoph WITTMANN.
Application Number | 20170350875 15/536798 |
Document ID | / |
Family ID | 52144443 |
Filed Date | 2017-12-07 |
United States Patent
Application |
20170350875 |
Kind Code |
A1 |
VAN DUUREN; Jost ; et
al. |
December 7, 2017 |
Method and Measuring Device for Determination of the Growth Rate of
Biofilm
Abstract
A method for determination of the growth rate of biofilm (7)
using an electrical impedance analyses is disclosed. The method
comprises the steps of: bringing a culture medium fluid (3) in
contact to an electrode structure (4a, 4b), having biofilm (7)
grown within the fluid culture medium (3) with the biofilm (7)
arranged in distance to the electrodes structure (4a, 4b), so that
the fluid culture medium (3) is placed between the growing biofilm
(7) and the electrode structure (4a, 4b); measuring the impedance
of the electrodes structure (4a, 4b) over a monitoring time, and
determining the growth rate of the biofilm (7) as a function of the
reduction rate of the impedance values measured on the electrode
structure (4a, 4b).
Inventors: |
VAN DUUREN; Jost;
(Saarbruecken, DE) ; KARGE; Blanka; (Braunschweig,
DE) ; WITTMANN; Christoph; (Saarbruecken, DE)
; BROENSTRUP; Mark; (Braunschweig, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Helmholtz-Zentrum fuer Infektionsforschung GmbH |
Braunschweig |
|
DE |
|
|
Assignee: |
Helmholtz-Zentrum fuer
Infektionsforschung GmbH
Braunschweig
DE
|
Family ID: |
52144443 |
Appl. No.: |
15/536798 |
Filed: |
December 18, 2015 |
PCT Filed: |
December 18, 2015 |
PCT NO: |
PCT/EP2015/080540 |
371 Date: |
June 16, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/025 20130101;
G01N 2333/21 20130101; G01N 27/041 20130101; G01N 33/4836 20130101;
C12Q 1/02 20130101 |
International
Class: |
G01N 33/483 20060101
G01N033/483; C12Q 1/02 20060101 C12Q001/02; G01N 27/04 20060101
G01N027/04 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 18, 2014 |
EP |
14198747.9 |
Claims
1. A method for determination of the growth rate of biofilm using
an electrical impedance analysis, comprising: bringing a fluid
culture medium in contact with an electrode structure, growing
biofilm within the fluid culture medium with the biofilm arranged
at a distance relative to the electrode structure so that the fluid
culture medium is between the biofilm which is being grown and the
electrode structure, measuring impedance values of the electrode
structure over a monitoring time, and determining a growth rate of
the biofilm as a function of a reduction rate of the impedance
values measured on the electrode structure.
2. The method according to claim 1, wherein the determining step is
performed by scaling the measured impedance values and determining
the growth rate of the biofilm as a function of the reduction rate
of the scaled impedance value.
3. The method according to claim 2, wherein the step of scaling the
measured impedance values is performed with respect to related
reference impedance values measured on a same or similar electrode
structure without biofilm.
4. The method according to claim 3, further comprising the steps of
determining scaled cell index values as a function of the impedance
values measured at different frequencies and of related reference
impedance values without biofilm, and determining the growth rate
of the biofilm in proportion to a reduction rate of said scaled
cell index values.
5. The method according to claim 1 wherein the step of determining
the growth rate of the biofilm is performed as a function of a
reduction rate of a reactance portion of the measured impedance
values, wherein the impedance values comprise the reactance portion
and a resistance portion.
6. The method according to claim 1 wherein the step of determining
the growth rate of the biofilm is performed after expiration of a
specific growth time, the specific growth time being expired at the
earliest when a continuous reduction of the impedance values
measured on the electrode structure occurs.
7. The method according to claim 1 wherein the step of determining
the growth rate of biofilm is performed for species having an
ability of forming pellicles.
8. The method according to claim 1 wherein the biofilm comprises
Pseudomonas aeruginosa.
9. The method according to claim 1 further comprising the step of
characterizing substances added to the fluid culture medium for
their ability to modulate growth of the biofilm.
10. A measuring device for determining a growth rate of biofilm,
comprising: at least one electrode structure, an impedance
measuring unit electrically connected or connectable to the
electrode structure for measuring impedance of the electrode
structure, and at least one receptacle for storing a fluid culture
medium, wherein the at least one receptacle is arranged for being
coupled to the electrode structure such that the fluid culture
medium is able to contact the electrode structure, an evaluation
unit arranged for determining the growth rate of biofilm located or
locatable at a distance from the electrode structure such that the
fluid culture medium is between the electrode structure and the
biofilm being grown, and wherein the evaluation unit evaluates
growth of the biofilm as a function of a reduction rate of the
impedance values measured on the electrode structure.
11. The measuring device according to claim 10, wherein the at
least one of receptacle comprises a chamber having a closed bottom,
and the electrode structure is arranged at the bottom of the
chamber in an internal space of the chamber.
12. The measuring device according to claim 10, wherein the at
least one receptacle comprises an upper chamber, a lower chamber,
and a microporous membrane between the upper chamber and the lower
chamber, and wherein the electrode structure is arranged adjacent
to the microporous membrane in an interconnection area of the upper
chamber and the lower chamber, and wherein the upper chamber is
configured to store the fluid culture medium with the biofilm
growing in the upper chamber.
13. The measuring device according to claim 10 wherein the
electrode structure comprises a pair of comb-type finger
electrodes, wherein fingers of the pair of comb-type finger
electrodes are alternately arranged adjacent and at a distance from
each other such that adjacent fingers extend in opposite directions
from each other.
14. The measuring device according to claim 10 wherein the at least
one receptacle includes a plurality of receptacles, wherein each
receptacle of the plurality of receptacles comprises a pair of
electrodes having a first electrode and a second electrode, and
wherein at least two of the first electrodes are electrically
interconnected to each other, and wherein the second electrodes are
individually controllable by the impedance measuring unit.
15. The measuring device according to claim 10 wherein the
evaluation unit is arranged for determining the growth rate of the
biofilm by scaling the measured impedance values and determining
the growth rate of the biofilm as a function of the reduction rate
of the scaled impedance value.
16. A method for characterizing substances for their ability to
modulate growth of a biofilm, comprising: growing the biofilm in a
measuring device which comprises at least one electrode structure,
an impedance measuring unit electrically connected or connectable
to the electrode structure for measuring impedance of the electrode
structure, and at least one receptacle for storing a fluid culture
medium, wherein the at least one receptacle is arranged for being
coupled to the electrode structure such that the fluid culture
medium is able to contact the electrode structure; and evaluating
the biofilm with an evaluation unit arranged for determining the
growth rate of the biofilm located or locatable at a distance from
the electrode structure such that the fluid culture medium is
between the electrode structure and the biofilm being grown, and
wherein the evaluation unit evaluates growth of the biofilm as a
function of a reduction rate of the impedance values measured on
the electrode structure.
17. The method of claim 7 wherein said species comprises a
pel-genotype.
Description
[0001] The invention relates to a method for determination of the
growth rate of biofilm using an electrical impedance analysis.
[0002] The invention further relates to a measuring device for
determining the growth rate of biofilm, said measuring device
comprising: [0003] at least one electrode structure; [0004] an
impedance measuring unit in electrical connection to the electrode
structure being provided for measuring the impedance of the
electrode structure, and [0005] at least one receptacle for storing
fluid culture medium, wherein the receptacle is arranged for being
coupled to the electrode structure such that the fluid culture
medium is able to contact the electrode structure.
[0006] The use of impedance analysis for monitoring microbiological
effects is well known.
[0007] K. Sachsenheimer, L. Pires, M. Adamek, Th. Schwartz and B.
E. Rapp: Monitoring Biofilm Growth using a Scalable Multichannel
Impedimetric Biosensor, in: 15th International Conference on
Miniaturized Systems for Chemistry and Life Sciences, Oct. 2-6,
2011, Seattle, Wash., USA, pages 1968 to 1970 discloses a
multichannel electrochemical impedance spectroscopy (EIS) based
biosensor that allows the monitoring of biofilm growth. The
bacterial strains (Pseudomonas aeruginosa, Stenotrophomonas
maltophilia) were monitored for up to 96 hours with the biofilm
directly growing on an electrode structure. Said electrode
structure comprises a working electrode and a counter electrode on
a substrate. The biofilm growth on the electrode structure hinders
the charge transfer between the electrodes and therefore increases
the measured impedance. The impedance increases over time because
of a biofilm growing on the electrode surface. Two electrodes are
used to compensate drift effects, with the measurement electrode
probe carrying the bacteria and a reference electrode being only
exposed to feeding medium.
[0008] WO 2005/047482 A2 and WO 2005/077104 A2 disclose a real time
electronic cell sensing system comprising an electrode structure
and a plurality of receptacles placed on top of the electrode
structure. Two or more electrode arrays are fabricated on a
non-conducting substrate. The substrate has a surface suitable for
cell attachment or growth. The cell attachment or growth on said
substrate results in a detectable change in impedance between the
electrode structures within each electrode array. For measurement
of the cell-substrate impedance, an impedance analyzer is connected
to the connection pads of the substrate for measuring the impedance
values at specific frequencies.
[0009] The impedance is a complex value comprising the ohmic
resistance and the reactance. The reactance is the imaginary part
of the impedance and provides the value of the capacitance of the
electrode structure. Cell Index values are calculated from the
measured impedance data. The dimensionless cell index measures the
relative change in the electrical impedance at certain frequency
(fn). The Cell Index at a given time point t (CI(t)) is calculated
as follows:
CI ( t ) = R ( f n , t ) - R ( f n , t 0 ) Z n ##EQU00001##
[0010] where
[0011] f.sub.n is the frequency that impedance measurement is
carried out,
[0012] R(f.sub.n, t) is the measured impedance at frequency f.sub.n
at time point t,
[0013] R(f.sub.n, t.sub.0) is the measured impedance at frequency
f.sub.n at time point t.sub.0, usually t.sub.0 is the time when the
background is measured),
[0014] Z.sub.n is the corresponding frequency factor of
f.sub.n.
[0015] For example, the xCELLigence.RTM. system, which is available
from the company ACEA Biosciences, Inc. USA, measures impedance at
three discrete frequencies, i.e., f1=10 kHz, f2=25 kHz, and f3=50
kHz. The corresponding frequency factors are Z1=15 Ohm, Z2=12 Ohm,
and Z3=10 Ohm, respectively.
[0016] The resistance and reactance part of the cell index can be
obtained by mathematical transformations. In another example, the
cell index can be calculated at each measured frequency by dividing
the resistance value and/or the reactance value of the electrode
arrays when cells are present on, or attached to the electrodes by
the baseline resistance and/or reactance. Hereby, the maximum value
in the reactance ratio over the frequencies spectrum can be found
or determined. Alternatively a specific value, e.g. the value 1 or
the measured value at the start of the experiment, can be
subtracted from the value in the reactance ratio.
[0017] Further examples for determining the cell index are
disclosed in the references WO 2005/047482 A2 and WO 2005/077104 A2
cited above.
[0018] WO 2004/010102 A2 discloses an impedance-based apparatus for
analyzing cells and particles comprising an upper chamber adapted
to receive and retain a cell sample, a lower chamber having at
least two electrodes, and a biocompatible porous membrane having a
porosity sufficient to allow cells to migrate therethrough. The
cells migrating to the lower chamber attach to the electrodes. In
another example the microporous membrane and the electrode
structure is placed between the upper and lower chamber. Again
cells are attaching to the electrode structure and growing on the
electrode structure. With increasing cell numbers, the impedance at
specific frequencies and the calculated cell index is increasing.
Thus, proportionally the capacity decreases with increasing cells
adhering on the electrode structure.
[0019] Said disclosed cell monitoring apparatus is commercial
available under the trademark xCELLigence.RTM. RTCA with CIM-Plates
and E-plates. Said device is available from the company ACEA
Biosciences, Inc. USA.
[0020] An object of the present invention is to provide an improved
method and measuring device for the determination of the growth
rate of biofilm.
[0021] The object is achieved by the method according with the
features of claim 1 and the measuring device comprising the
features of claim 9. Preferred embodiments are disclosed in the
dependent claims.
[0022] According to the present invention, the growth rate of the
biofilm is determined as a function of the reduction rate of the
impedance values measured on the electrode structure with the
biofilm not adhering to the electrode structure.
[0023] Therefore, the method comprises steps of: [0024] bringing a
fluid culture medium in contact to an electrode structure, [0025]
having a biofilm grown within the fluid culture medium with the
biofilm arranged in distance to the electrode structure, so that
the fluid culture medium is placed between the growing biofilm and
the electrode structure, [0026] measuring the impedance of the
electrode structure over a monitoring time, and [0027] determining
the growth rate of the biofilm as a function of the reduction rate
of the impedance values measured on the electrode structure.
[0028] While the prior art proposes the determination of the
surface impedance on an electrode structure having a pair of
electrodes with the biofilm growing on the electrodes, the present
invention proposes determination of the biofilm growth rate as a
function of the dielectric change of the capacitive structure
between the biofilm and the electrodes. Due to an increasing
dielectric constant of the doublecharged layer caused by biofilm
growth, the capacitance increases. This is achieved by placing the
culture medium in a fluid form between the electrode structure and
the biofilm, so that the biofilm floats on the culture medium and
does not directly contact the electrode structure. As a result, the
impedance values measured on the electrode structure shows a
reduction in relation to the growing rate of the biofilm. When
adhering the biofilm on the electrode structure according to the
prior art, the impedance values increases with growing biofilm.
[0029] The use of a fluid for the culture medium instead of a gel
allows the biofilm to float on top of the fluid culture medium in
distance to the electrode structure.
[0030] Determining the biofilm formation rate as a function of a
reduction rate of the impedance values of the electrode structure
with the fluid culture medium being placed between the electrode
structure and the biofilm has the advantage of a very low standard
deviation and an increased distinctiveness of the measurement
results with reproducible values. A high number of probes can be
measured in parallel within a short time frame.
[0031] In a preferred embodiment, the measured impedance values are
scaled and the growth rate of the biofilm is determined as function
of the reduction rate of the scaled impedance values. By scaling
the measured impedance values, normalized impedance values are
achieved, so that the influence of the electrical impedance of the
measurement device is faded out.
[0032] The scaling of the measured impedance values is preferably
performed with respect to related reference impedance values
measured on the same or similar electrode structure without
biofilm. Thus, the measured impedance values on an electrode
structure with biofilm are scaled with the related reference
impedance value for the same or similar electrode structure without
biofilm. The related reference impedance values can be achieved for
example before inserting biomass in the fluid culture medium at a
first step. Later, after inserting biomass into the fluid culture
medium the impedance values are measured and compared with the
referenced impedance values measured in the first step before said
second step with biomass.
[0033] Another option is to provide similar electrode structures,
wherein the impedance values are measured on samples with biomass
in parallel to samples without biomass and biofilm growing due to
biomass within the fluid culture medium, in order to obtain biomass
impedance values and related reference impedance values.
[0034] Scaling can be taken place by calculating the quotient
between the measured biomass impedance values and the related
reference impedance values or by subtracting both values.
[0035] In a most preferred embodiment, scaled cell index values are
determined as a function of the impedance values measured at
different frequencies and of related reference impedance values
without biofilm. The impedance values for the different frequencies
obtained at the same timepoint is then taken into account. For
example, the maximum of the impedance values can be considered. The
growth rate of the biofilm is determined in proportion to the
reduction rate of the said scaled cell index values.
[0036] For example, the cell index values provided by the prior art
xCELLigence.RTM. RTCA available from the company ACEA Biosciences,
Inc. can be used for determining the growth rate of the
biofilm.
[0037] A possible variant of the method provides that the growth
rate of the biofilm is determined as a function of the reduction
rate of the reactance portion of the measured impedance value. The
measured impedance value comprises a reactance portion and a
resistance portion. In this preferred embodiment, only the
reactance portion of the impedance is considered for determination
of the growth rate of the biofilm. The reactance is the imaginary
part of the complex impedance providing a value for the capacitance
of the electrode structure and the fluid culture medium placed
adjacent to the electrode structure with the biofilm growing in the
fluid culture medium in distance to the electrode structure. With
growing biofilm, the dielectric properties of the biofilm are
changing, thus resulting in an increasing capacitance. In the prior
art with cells adhering to the electrode structure, the surface
impedance is effective with growing cells causing an increasing
impedance and ohmic resistance.
[0038] Considering only the reactance portion of the impedance has
the advantage of highly reliable measurement values with very low
standard deviation and increased distinctiveness most likely due to
the fact that only the dielectric behaviour of the biofilm is
evaluated for monitoring the biofilm growth rate.
[0039] In a preferred embodiment, the growth rate of the biofilm is
determined after expiration of a specific growth time. Said
specific growth time expires at the earliest when a continuous
reduction of the impedance values measured of the electrode
structure occurs. The specific growth time is preferably set to a
time when biofilm starts to grow significantly, e.g. a time when
pellicle is formed.
[0040] Thus, the change of the impedance values measured on the
electrode structure for at least one frequency is monitored over
the monitoring time. It had been experimentally noted, that in a
first phase, the impedance values and in particular the cell index
or reactance first significantly decreases within a short time of
approximately 1 to 4 hours. Then, the values are increasing until
expiration of the specific growth time. The growth time depends on
the biomass and the specific experimental environment. After
expiration of the specific growth time, a continuous reduction of
the impedance values occurs. After determination of such a
continuous reduction of the measured impedance values or related
scaled values or cell index values, the expiration of the growth
time is determined. Then, the biofilm growth rate can be determined
within the time window of the monitoring time said considered time
window being after expiration of the growth time. A mathematical
function that describes the biofilm formation can be defined, based
on which the slope of the reduction (which is equal to the
directional cell index) can be calculated within this period. For
example, the specific growth time can be set to a value in a range
of 30 to 40 hours and preferably 30 to 35 hours after placing fluid
culture medium with biomass or growing biofilm in a receptacle
arranged on an electrode structure. The growth rate of the biofilm
is then determined within a measurement time of for example 5
hours.
[0041] The method for determining the growth rate of biofilm makes
most preferably use of the xCELLigence.RTM. Real-time cell analyzer
of ACEA Biosciences, Inc. or with another suitable device
comprising an electrode structure and at least one receptacle
provided for placing a fluid culture medium with biomass for
growing biofilm on the electrode structure according on the prior
art or of biofilm arranged in distance to the electrode structure.
The electrode structure comprises at least one measurement
electrode and one counter electrode. The measuring device comprises
an impedance measuring unit for applying a measurement signal with
at least one specific frequency on both electrodes of the electrode
structure in order to determine the complex impedance on the
electrode structure and in particular the related reactance.
[0042] Most preferably, the E96 plates provided for the
xCELLigence.RTM. Real-time cell analyzer are applicable for the
determination of the growth rate of biofilm floating on the fluid
cell medium.
[0043] The impedance values, in particular the reactance, are
further evaluated by use of an evaluating unit in order to perform
the above mentioned method. The evaluation unit can be for example
a data processing unit, e.g. microcontroller, microprocessor or
computer having a computer program for performing the evaluation
steps as described above. The evaluation unit can also be a field
programmable array FPGA providing a hardware device for performing
the evaluation steps.
[0044] The invention is described by use of exemplary embodiments
with the enclosed drawings. It shows:
[0045] FIG. 1--Block diagram of a first embodiment of a measuring
device for determining the biofilm growth rate;
[0046] FIG. 2--Block diagram of a second embodiment of a measuring
device for determining the biofilm growth rate comprising a
microporous membrane;
[0047] FIG. 3--Diagram of crystal violet (CV) by monitoring
absorption at 550 nm staining measurements of biofilms formed by
PA14 (wild type WT, .DELTA.pelA, and .DELTA.pqsA) in a fluid
culture medium LB at various timepoints (FIG. 3A), compared to the
cell index measurements (FIG. 3B) of these strains under equal
conditions;
[0048] FIG. 4--Diagram of the cell index over the time for
determining the PA14 biofilm growth rate in a fluid culture medium
LB with .DELTA.pelA, wild type WT and .DELTA.pqsA;
[0049] FIG. 5--Diagram of PA 14 biofilms determined after 43 hours
with crystal violet by monitoring absorption at 620 nm;
[0050] FIG. 6--Diagram of the RK cell index per hour for PA14
biofilms determined by impedance spectroscopy.
[0051] FIG. 7a)--Diagram of the cell index over the time for a
control experiment after transplantation of biofilm, the addition
of PBS and the LB medium as negative control.
[0052] FIG. 7b)--Diagram of the cell index over the time for an
experiment where paraffin was also added in different amounts
(10-100 .mu.L) to wells (B) to mimic pellicle biofilm, given
paraffin has no dielectric constant its presence showed no effect
on the capacitance.
[0053] FIG. 8a-c)--Diagram showing the impact of ciprofloxacin on
biofilm formation of PA14.
[0054] FIG. 9a-c)--Diagram showing the impact of tobramycin on
biofilm formation of PA14.
[0055] FIG. 10a-c)--Diagram showing the impact of meropenem on
biofilm formation of PA14.
[0056] FIG. 1 shows a block diagram of a measuring device 1 for
determining the growth rate of biofilm. The measuring device 1
comprises a plurality of receptacles 2a, 2b provided for storing a
fluid culture medium 3. Each receptacle 2a, 2b comprising an
electrode structure 4a, 4b. In the exemplary embodiment, each
electrode structure 4a, 4b is placed at the bottom in the interior
space of a chamber 5 formed by a related receptacle 2a, 2b. The
receptacles 2a, 2b are placed such, that the fluid culture medium 3
is able to contact the related electrode structure 4a, 4b, wherein
one electrode acts as measurement electrode 6a and the other as
counter electrode 6b. The electrode structures 4a, 4b each are
formed by a pair of comb-type finger electrodes. Each comb-type
finger electrode comprising a plurality of fingers extending in the
same direction and being placed adjacent to each other like a comb.
The pair of comb-type finger electrodes are interdigitating
arranged such that the fingers of the pair of comb-type finger
electrodes being alternately placed adjacent and in distance from
each other such that the adjacent fingers extending in opposite
directions from each other.
[0057] When injecting biomass into the fluid culture medium 3, a
biofilm 7 is growing within the respective chamber 5 and the
biofilm 7 is floating on top of the fluid culture medium 3.
[0058] Other than in the prior art, by use of fluid culture medium
3 and having the biofilm 7 floating on top of the fluid culture
medium 3, the biofilm 7 will not adhere to the electrode structure
4a, 4b.
[0059] For all exemplarily described embodiments of the invention
and other variants, the impedance of the electrode structures 4a,
4b are each measured by use of an impedance measuring unit 8 in
electrical connection to the electrode structures 4a, 4b. In order
to measure the impedance, a measuring signal, e.g. a AC sinus wave,
is applied to a pair of electrodes of the selected electrode
structure 4a, 4b, i.e. the measurement electrode 6a and the counter
electrode 6b, and the signal amplitude and phase is then measured.
For example when keeping the current constant, the voltage
amplitude and phase between the measurement signal and the measured
signal will vary as a function of the capacitance. When applying a
measuring signal with constant voltage, the current amplitude and
the phase angel will vary accordingly. Thus, the impedance
measuring unit 8 is arranged to measure the impedance value of a
selected electrode structure 4a, 4b. The impedance measuring unit 8
may have a multiplexer for selecting one of the plurality of
electrode structures 4a, 4b after the other in order to perform
fast impedance measurement for a plurality of receptacles 2a, 2b
and their related electrode structures 4a, 4b. The impedance is the
sum of the ohmic resistance and the complex reactance:
Z=Z.sub.R+Z.sub.C=R+1/j.omega.C
where .omega. is the frequency of the measurement signal, C the
capacity and R the ohmic resistance.
[0060] The capacity C is related to the reactance with a kind of
exponential function. The capacity C is linear proportional to In
(Z).
[0061] The growth rate of biofilm 7 floating on the fluid culture
medium 3 is a function of the reduction rate of the reactance and
the increase rate of the capacitance C.
[0062] Therefore, the measuring device 1 comprises an evaluation
unit 9 arranged for determining the growth rate of biofilm 7 being
located in distance to the electrode structure 4a, 4b with the
fluid culture medium 3 placed between the electrode structure 4a,
4b and the biofilm 7 as a function of the reduction rate of the
impedance values measured on the related electrode structure 4a, 4b
by use of the impedance measuring unit 8.
[0063] FIG. 2 shows a schematic block diagram of a second
embodiment of a measuring device 1. In this second embodiment, the
receptacles 2a, 2b each comprising an upper chamber 5a and a lower
chamber 5b. The electrode structure 4a, 4b each is fixedly mounted
at the bottom of the related lower chamber 5b. The upper chambers
5a are provided for storing the fluid culture medium 3a, 3b and for
being placed in the related lower chamber 5b after inserting the
fluid culture medium 3 into the related upper chamber 5a and
optionally also into the lower chamber 5b. The upper chambers 5a
are closed with a microporous membrane 10 at their bottom. When
injecting biomass into the fluid culture medium 3, a biofilm 7 is
growing within the upper chamber 5a. The biofilm 7 is floating on
top of the fluid culture medium 3 and cells are hindered to reach
the electrode structure 4a, 4b at the bottom of the respective
lower chamber 5b and to adhere to said electrode structure 4a,
4b.
[0064] Thus, when inserting the upper chamber 5a in the
corresponding lower chamber 5b, the fluid culture medium 3 will be
placed on top on the electrode structure 4a and 4b and the biofilm
7 will grow in distance to the electrode structure 4a, 4b. The
microporous membrane 10 has the effect of acting against cells
adhering to the electrode structure 4a, 4b, so that interfering
effects to the measurement result are reduced. The pore size of the
microporous membrane 10 is, for this purpose, preferably smaller
than the mean cell diameter.
[0065] FIG. 3 shows a diagram of the cell index for biofilms of
Pseudomonas aeruginosa measured by use of the impedance
spectroscopy device xCELLigence.RTM. Real-time cell analyzer
according to the prior art (FIG. 3B), in comparison to a standard
method for biofilm quantification that is based on staining of the
biofilm with crystal violet (FIG. 3A). The diagram shows that the
characteristic change in cell index occurs at time periods (ca.
30-45 h) that are featured by significant growth of biofilm.
[0066] The experiment was repeated for a number of n with n=12 for
the well type WT and pelA and pqsA mutants. For the medium control
n was equal to 8. .DELTA.pelA is a mutant of the wild type WT
without the pelA gene, showing only planktonic growth of biofilm.
.DELTA.pqsA is a deletion mutant showing growth of biofilm with an
increased capacity as compared to the wild type WT. .DELTA.pelA
grown within the fluid culture medium provides an almost horizontal
line without significant reduction. Thus, there is no biofilm
growing within this sample. With the .DELTA.pelA probe it is also
verified that the method according to the present invention is not
sensitive for planktonic cells.
[0067] In the example according to diagram of FIG. 3, the cell
index is calculated based upon the scaled impedance.
[0068] FIG. 4 shows a diagram of the cell index over the time for a
plurality of probes measured with the xCELLigence.RTM. Real-time
cell analyzer for biofilm of Pseudomonas aeruginosa. The biomass
Pseudomonas aeruginosa had been measured as an exemplary biomass.
Eventually also other strains can be analyzed. The 96 E-Plate had
been used for a measuring time of 43 hours. The fluid culture
medium LB had been introduced into the receptacles together with
wild type (WT) PA14, .DELTA.pelA deletion mutant or .DELTA.pqsA,
respectively.
[0069] When placing a fluid culture medium LB between the electrode
structure 4a, 4b and the biofilm 7, the value of the cell index
shows a fast decrease within a starting phase, a following increase
and, after expiration of the specific growth time, e.g. of ca. 20
hours, an almost linear decrease. A significant linear reduction of
the negative cell index occurs after about 35 hours and more
preferably in the time frame of the 37 hours to 42 hours. Within
this time frame of 37 hours to 42 hours, the directional cell index
RK=(cell index[t2]-cell index[t1])/(t2-t1) is decreasing. Thus, for
such a short time frame, the value of the cell index change
provides the directional cell index RK, which corresponds to the
linear relation between the capacitance and the reactance having
the general form of a straight line. Optionally, and in particular
in case of evaluating longer time frames, the growth rate of
biofilm can be calculated as function of the natural logarithm of
the cell index, e.g. by the formula RK=In(cell index[t2]-cell
index[t1])/(t2-t1).
[0070] The directional cell index RK has, in this example, a value
of -7.25E-03/h.+-.7.56E-04 for the biofilm growth rate of WT.
[0071] For the biofilm growth rate of .DELTA.pqsA, the directional
cell, index RK has a value,of -1.07E-02/h.+-.1.01E-03.
[0072] The measurements had been taken for a number of 12
samples.
[0073] The biofilm growth rate can be determined according the
impedance measurement with fluid culture medium placed between the
electrode structure and the biofilm. The biofilm growth rate is
proportional to the directional coefficient of the capacitance,
e.g. the gradient of (Z) or (cell index) when considering the value
of Z or the related cell index. For longer time frames linearizing
by use of the natural, logarithm might be necessary, so that
biofilm growth rate is proportional to the gradient of In (Z) or In
(cell index).
[0074] Thus, the biofilm growth rate is directional proportional
to
(cell index(t2)-(cell index (t1)).
and for longer, time frames between t1 and t2 by use of the natural
logarithm
In(cell index(t2)-cell index (t1)).
[0075] The variable t1 is the first time of the measurement window
after a specific growth time e.g. 37 hours, while t2 is the end
time of measuring time, e.g. 42 hours.
[0076] The cell index or impedance curve shows a continuous
reduction within this time measurement time frame t1 to t2.
[0077] FIG. 5 is a diagram of various types of PA14 biofilms
measured by use of the 96-Well-Plate after 43 hours by use of the
fluid culture medium LB with crystal violet analysis by monitoring
absorption at 620 nm.
[0078] It is obvious that compared to the wild type WT alone, the
addition of arginine (L-Arg) to the wild type WT inhibits the
growth of biofilm.
[0079] FIG. 6 shows a diagram of the RK cell indices obtained for
various types of PA14 biofilms grown with the fluid culture medium
LB by use of the impedance analysis with a 96-xCELLigence.RTM.
Well-Plate within the evaluation time of 37 to 42 hours.
[0080] For .DELTA.pqsA the highest negative RK cell index occurs,
which is related to the biofilm growth rate. The use of 0.4% or
0.8% Arginine (L-Arg) reduces the biofilm growth rate by about
1/2.
[0081] Adding 0.4% or 0.8% Arginine (L-Arg) to the wild type WT
results in reduction of the biofilm growth rate compared to the
wild type WT without the presence of L-Arg. Again the directional
cell index RK of about -0.004/h is directly proportional to the
biofilm growth rate.
[0082] The factor r2 provides information on the average
straightness of the line in the section considered for determining
the biofilm growth rate.
[0083] As can be seen when comparing the diagrams in FIG. 5 and
FIG. 6, the standard deviation can be significantly decreased and
the distinctiveness can be increased by impedance analysis of the
biofilm growth rate when measuring the reduction of the reactance
with fluid culture medium between the biofilm and the electrode
structure. The impedance analysis allows measuring of a plurality
of probes in parallel within short time.
[0084] The measuring device can also be provided in front of an
integrated circuit or integrated chip array.
[0085] In particular, the growth time of biofilm can be shortened
by reducing the volume of the fluid culture medium 3 (LB). Due to
the reduced volume cells reach earlier a threshold of cell density
where they are going to produce biofilm.
[0086] FIGS. 3 and 4 shows that the growth of biofilm does not
occur continuously but in different phases. The method for
measuring the biofilm growth when the capacitance increases and the
reactance decreases can not only be used for determining
Pseudomonas aeruginosa PA14 biofilm growth. Other classes of
biomaterials exhibit the same effect, in particular all ESKAPE
organisms. ESKAPE pathogens are Enterococcus faecium,
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa, and Enterobacter species.
[0087] When monitoring the different phases of biofilm growth with
their characteristic changes of the cell index curve over the
measurement time, it is possible to define suitable times for
taking probes of biomass from the receptacles in order to analyze
the probe as required. Thus, the cell index curve over the time
measured with the possible presence of biofilm floating on the
fluid culture medium and not significantly adhering to the
electrode structure provides an indicator for the appropriate
timing of samples to be taken from the biofilm.
[0088] In order to probe the specific effect of the pellicle
biofilm on the cell index, two experiments had been performed.
[0089] FIG. 7a) shows the result of the Cell index over the time
measured with the Xcelligence.RTM. system, where Biofilm was
transplanted to an adjacent receptacle with a metal loop to measure
the effect of its presence on the impedance spectroscopy (n=4)
compared to the addition of 10, 20, 40, and 80 .mu.l PBS buffer
(n=1)--the average value is shown--and no addition (LB medium,
n=8).
[0090] FIG. 7b) shows the result of the Cell index over the time
measured with the Xcelligence.RTM. system, where paraffin was also
added in different amounts (10-100 .mu.L) to wells (n=4) in order
to mimic pellicle biofilm. The results were compared to the sole LB
medium (n=8). Given paraffin has no dielectric constant its
presence showed no effect on the capacitance.
[0091] First, a pellicle biofilm obtained by growing PA14 in LB
medium for 72 h was transplanted, with a metal loop into wells
filled with LB medium. In control experiments, PBS buffer was added
to LB medium as shown in FIG. 7a).
[0092] Secondly, as shown in FIG. 7b), paraffin (10-100 .mu.l) was
added as an artificial substrate for comparison. Paraffin is
immiscible with water and therefore floats on top of the
hydrophilic medium due to its lower density. Because its chemical
composition consists of alkanes, paraffin doesn't have a dielectric
constant, in contrast to the pellicle biofilm of P. aeruginosa. For
both experiments, the Xcelligence.RTM. system was calibrated once
and paused before addition. It can be observed, that the slope of
decrease after transplantation of biofilm was steeper than that
obtained for an addition of PBS alone and the LB medium. This
effect cannot be explained by the addition of an immiscible layer
on top of the LB medium, as the addition of paraffin led to
unchanged slopes of cell index.
[0093] The impedance method has also been used to study the effect
of antibiotics on biofilm formation of P. aeruginosa PA14. The
results of the impact of ciprofloxacin (A), tobramycin (B), and
meropenem (C) on biofilm formation of PA14 are shown in FIGS. 8 to
10. As examples, the antibiotics ciprofloxacin (FIGS. 8a)-c)),
tobramycin (FIGS. 9a)-c)) and meropenem (FIGS. 10a)-c)) have been
tested. All three antibiotics changed the impedance curves in a
concentration-dependent manner, indicating effects on biofilm
formation. Interestingly, impedance spectroscopy could monitor an
effect on biofilm formation at antibiotic concentrations that did
not impact planktonic growth at the same period of time. For
example, the addition of 0.13 .mu.g/mL ciprofloxacin clearly
changed the impedance curve reflecting altered biofilm formation,
although planktonic growth was not impaired at this concentration.
At a concentration of 0.25 .mu.g/mL, steady biofilm formation was
not observed anymore, but planktonic growth was still possible.
[0094] The effect on impedance was quantified by three parameters
at varying antibiotic concentrations:
[0095] The first parameter is the time of onset of a linear
decrease of the cell index, (FIGS. 8a), 9a) and 10a)).
[0096] The second parameter is the duration of a linear decrease of
the cell index. This duration was determined by a linear trendline
that was fitted to the data with an r.sup.2 value of 0.97 or
higher. In the graphs of FIGS. 8a), 9a) and 10a), the black
vertical bars represent the time period over which the biofilm
formation rate was measured to be steady, based on a straight line
assigned with an r.sup.2 of at least 0.97 (n=3 or 4).
[0097] The third parameter was the cell index slope during that
time period (FIGS. 8b), 9b) and 10b)). These graphs show the
concentration dependency of the biofilm formation expressed in the
slope/ h of the cell index (n=3 or 4).
[0098] For ciprofloxacin and tobramycin, a shallower decline could
be observed at increasing concentrations, indicating an impaired
biofilm formation. For meropenem on the other hand, the increasing
cell index slope suggests that it induced biofilm formation at
concentrations between 0.063 and 0.25 .mu.g/ml; this effect
vanished at 0.5 .mu.g/ml. Especially at the highest tested
concentrations, all antibiotics led to later onsets of linear
declines of impedance; at the same time, the period of linear
decline was longer compared to untreated samples, indicating a
delayed and attenuated biofilm formation.
[0099] FIGS. 8c) to 10c) show the result of the control experiment
by measuring the OD600 value over the time with UV spectroscopy.
OD600 indicates the absorbance, or optical density, of a sample
measured at a wavelength of 600 nm and indicates the concentration
of cells in a liquid. These graphs of FIGS. 8c), 9c) and 10c)
represent growth curves of PA14 at various concentrations over time
(n=4). At t=0 the OD600 was installed at 0.1.
[0100] The method allows determining the effect of other classes of
modulators, for example [0101] standard of care drugs used for the
treatment of P. aeruginosa, including aminoglycosides (e.g.
Amikacin, Tobramycin) Fluorochinolones (e.g. Ciprofloxacin,
Levofloxacin, Ofloxacin) beta-lactam antibiotics (e.g. Meropenem,
Doropenem, Imipenem, Piperacillin, Cefepime); [0102] Compounds that
have shown activity against P. aeruginosa in vitro and/or in vivo
models (e.g. POL7080, Eravacyclin, Omadacycline, Plazomicine,
Ceftazidime-Avibactam, Ceftolozane/Tazobactam); [0103] tool
compounds with known positive or negative effects on of biofilm
growth (e.g. arginine and casamino acid); [0104] agonists and
antagonists of quorum sensing (e.g. from the classes of AHL- or
PQS-analogs); [0105] Virulence factors (e.g. compounds interfering
with secretion systems like anti-PcrV antibodies).
[0106] The results of the method for determining the growth of
biofilm can be used to present online phenotypical effects of the
biofilm growth. Thus, molecular changes can be made visible that
are related to [0107] Membrane composition [0108] Metabolites
[0109] Proteome [0110] Transcriptome [0111] Fluxome.
* * * * *